Monoamine reuptake is a major mechanism for regulating extraneuronal monoamine levels and terminating synaptic transmission. Reuptake is mediated by plasma membrane transporters that are the primary targets for psychostimulants such as cocaine, methamphetamine and MDMA ("Ecstasy"), as well as for therapeutic drugs such as fluoxetine (Prozac), sibutramine (Meridia), bupropion (Wellbutrin) and methylphenidate (Ritalin). These agents block reuptake, resulting in elevated extraneuronal monoamine levels and enhanced postsynaptic responses. Recent evidence demonstrates that transporters are subject to acute regulation by cellular signaling pathways. Transporter regulation is coupled to dynamic changes in transporter cell-surface presentation, suggesting that membrane trafficking is fundamental to transporter homeostasis and regulation. However, the cellular and molecular mechanisms governing transporter regulation and trafficking are not yet defined. Given the pronounced effect pharmacological transporter blockade exerts on synaptic transmission, it is highly likely that transporter sequestration also has significant downstream effects on neuronal signaling. Moreover, modulation of transporter availability is certain to have significant impact on the efficacy of psychoactive drugs. The major goals of this project are to elucidate the cellular and molecular mechanisms mediating acute transporter regulation and trafficking. This investigative line will be pursued by testing the following hypotheses: (1) Transporters undergo constitutive internalization and recycling, and (2) transporter regulation is achieved by altering transporter trafficking kinetics. These hypotheses are based on strong preliminary data that the dopamine transporter (DAT) undergoes constitutive endosomal trafficking and that protein kinase C (PKC) activation directly alters DAT trafficking. The proposed hypotheses will be tested by directly analyzing basal and regulated transporter trafficking kinetics in cell lines. Intrinsic domains mediating basal and PKC-regulated DAT trafficking will be identified using molecular truncation and mutagenesis approaches. It is expected that these approaches will provide a clear and comprehensive picture of the mechanisms underlying acute transporter modulation. Such results are expected to have a significant impact on future therapeutic strategies aimed at monoamine-related drug abuse and mental illnesses. Moreover, the outcomes will greatly improve our understanding of the factors contributing to monoamine availability and signaling in the brain.